ZSWIM4 inhibition improves chemosensitivity in epithelial ovarian cancer cells by suppressing intracellular glycine biosynthesis

Background Zinc finger SWIM-type containing 4 (ZSWIM4) induces drug resistance in breast cancer cells. However, its role in epithelial ovarian cancer (EOC) remains unknown. In this study, we aimed to investigate the clinical significance of ZSWIM4 expression in EOC and develop new clinical therapeutic strategies for EOC. Methods ZSWIM4 expression in control and EOC tumor tissues was examined using immunohistochemistry. Lentiviral transduction, Cell Counting Kit-8 assay, tumorsphere formation assay, flow cytometry, western blotting, and animal xenograft model were used to assess the role of ZSWIM4 in chemotherapy. Cleavage Under Targets and Tagmentation (CUT&Tag) assays, chromatin immunoprecipitation assays, and luciferase reporter assays were used to confirm FOXK1-mediated upregulation of ZSWIM4 expression. The mechanism by which ZSWIM4 inhibition improves chemosensitivity was evaluated using RNA-sequencing. A ZSWIM4-targeting inhibitor was explored by virtual screening and surface plasmon resonance analysis. Patient-derived organoid (PDO) models were constructed from EOC tumor tissues with ZSWIM4 expression. Results ZSWIM4 was overexpressed in EOC tumor tissues and impaired patient prognoses. Its expression correlated positively with EOC recurrence. ZSWIM4 expression was upregulated following carboplatin treatment, which, in turn, contributed to chemoresistance. Silencing ZSWIM4 expression sensitized EOC cells to carboplatin treatment in vitro and in vivo. FOXK1 could bind to the GTAAACA sequence of the ZSWIM4 promoter region to upregulate ZSWIM4 transcriptional activity and FOXK1 expression increased following carboplatin treatment, leading to an increase in ZSWIM4 expression. Mechanistically, ZSWIM4 knockdown downregulated the expression of several rate-limiting enzymes involved in glycine synthesis, causing a decrease in intracellular glycine levels, thus enhancing intracellular reactive oxygen species production induced by carboplatin treatment. Compound IPN60090 directly bound to ZSWIM4 protein and exerted a significant chemosensitizing effect in both EOC cells and PDO models. Conclusions ZSWIM4 inhibition enhanced EOC cell chemosensitivity by ameliorating intracellular glycine metabolism reprogramming, thus providing a new potential therapeutic strategy for EOC. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-024-04980-8.


Introduction
Among all gynecological malignancies, ovarian cancer (OC) has the highest mortality rate [1].Among all OC types, epithelial ovarian cancer (EOC) is the most common, accounting for over 90% of all cases [2].Over the past three decades, platinum-based therapy has represented the most administered treatment for patients with EOC [3].However, owing to disease heterogeneity, many patients experience recurrence, portending an extremely low survival rate [4].The mechanisms of EOC chemoresistance are diverse, including increased intracellular drug pumping [5], deactivation of tumor suppressor gene (retinoblastoma and phosphatase and tensin homolog) transcription [6], and reverse mutations in breast cancer (BRCA )1 and BRCA2 [7].However, the benefits of current treatment strategies for patients with EOC remain limited, with a poor overall prognosis.Therefore, an urgent need exists for the identification of molecular markers affecting recurrence in patients with EOC and the development of novel therapeutic targets to improve their clinical prognosis.
Zinc finger switching (SWI)2/Sucrose NonFermenting (SNF)2 and MuDR (SWIM) type-containing (ZSWIM) family members are named due to the uniqueness of their structural domains [8].Reports suggest that ZSWIM proteins have strong regulatory functions in cells, whether they bind to deoxyribonucleic acid (DNA) to regulate gene transcription [9] or play a role in ubiquitination to affect target protein expression [10,11].Increased ZSWIM4 expression is correlated with the high clinical stage of colon adenocarcinoma [12], and activated transforming growth factor beta (TGF-β) signaling results in the upregulation of ZSWIM4 in breast cancer cells [13].Our previous study showed that ZSWIM4 could mediate resistance to apoptosis induced by a non-receptortype tyrosine-protein Janus kinase 2 (JAK2) inhibitor by upregulating vitamin D receptor levels in breast cancer cells.Furthermore, ZSWIM4 knockdown could counteract resistance to JAK2 inhibitors [14].These findings indicate that high ZSWIM4 expression in tumor cells could contribute to drug resistance.However, its function in ovarian malignancy has rarely been studied.Hence, clarifying the specific role of ZSWIM4 in EOC is imperative for the clinical treatment of this disease.
The process by which cells promote growth and proliferation regulating their metabolism is known as metabolic reprogramming [15].Tumor cells adaptively change their metabolic processes to meet their required proliferation capacity; therefore, metabolic reprogramming is considered a new tumor marker [16].Recently, increasing evidence has shown that repression of aberrant metabolism within tumor cells enhances EOC cell chemosensitivity.For example, inhibiting fatty acid oxidation enhances the therapeutic efficacy of platinum drugs in EOC [17].Moreover, active amino acid metabolism in EOC cells is crucial for maintaining stem cell-like characteristics [18] and drug resistance [19].Meanwhile, one-carbon unit pathway metabolism reportedly plays an important role in cancer treatment [20].Targeting the key enzymes involved in serine and glycine biosynthesis elicits a synergistic effect with Olaparib in EOC cells [21].However, the abnormal metabolism of reduced glutathione (GSH) in EOC cells is a vital driver of platinum resistance [22], and GSH inhibition enhances EOC cell sensitivity to platinum drugs [23].Although several studies have shown that metabolic reprogramming contributes to the therapeutic resistance of EOC, the specific drivers and mechanisms underlying its regulation remain unknown.Uncovering the molecular mechanisms by which tumor cells regulate metabolic reprogramming to exhibit chemoresistance could provide a more definitive theoretical basis for overcoming EOC chemoresistance.
In this study, we report that ZSWIM4 is highly expressed in EOC tumor tissues and is responsible for chemoresistance in EOC cells.Moreover, ZSWIM4 inhibition improves EOC cell chemosensitivity by regulating intracellular glycine metabolism.Collectively, our study provides a novel target for overcoming chemotherapy resistance in EOC cells.

Ethics statement
The acquisition and use of human specimens were in concordance with the International Ethical Guidelines for Research Involving Human Subjects, as stated in the Helsinki Declaration, and were approved by the Medical Ethics Committee of Guangzhou Women and Children's Medical Center (2022416B01).All the tissue samples were collected after obtaining signed informed consent from the participants.The use of ovarian cancer tissue microarray (TMA) was approved by the Ethics Committee of Shanghai Outdo Biotech Company (SHYJS-CP-1804010).All experiments involving animals were conducted according to the ethical policies and procedures approved by the Southern Medical University Animal Care and Use Committee (L-2020163).

Cell culture and sample collection
Three human EOC cell lines (OVCAR8, SKOV3 and ES-2) were obtained from Procell Life Science & Technology (Wuhan, China).Normal human ovarian surface epithelial cells (IOSE-80) were obtained from iCell Bioscience Inc. (Shanghai, China).The cells were tested and confirmed to be free of Mycoplasma.The cell passage number was no more than 20.OVCAR8 cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco).SKOV3 cells were cultured in McCoy's 5A (Gibco) plus 10% FBS.Carboplatin (CBP)-resistant SKOV3 cells (SKOV3/CBP) were generated from parental SKOV3 cells by treating them with gradually ascended concentrations of CBP.All cells were cultured at 37 °C with 5% CO 2 .
We collected 15 paraffin-embedded human EOC tissue sections and 15 paraffin-embedded corresponding normal sections, including 10 paired samples and two fresh human EOC tumor tissue samples, for this study.All samples were obtained from September 2021 to June 2023 from patients from Guangzhou Women and Children's Medical Center, Guangzhou, China diagnosed with EOC who had not received chemotherapy before surgery.The ovarian cancer tissue microarray (TMA) (Cat.HOv-aC151Su01) was provided by Outdo Biotech (Shanghai, China).Excluding the exfoliated samples and necrotic tissues, we included 117 cases of primary lesions in our analysis, including 114 cases of EOC and three cases of tumors from germ cells.Our analysis was based on the ZSWIM4 immunohistochemical score in the 114 cases.

Cell viability assay
For the cell viability assay, EOC cells were cultured at a density of 5,000 cells/well in 96-well plates and treated with test compounds for the indicated time points.In the experiment using CBP, physiological saline was used as the control treatment.The cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) (Cat.KGA317, Key-Gen, Nanjing, China).Construction and statistical analysis of the drug 50% maximal inhibitory concentration (IC 50 ) curves were carried out using GraphPad Prism ver.6.0.as previously described [14].Statistical analyses (mean ± standard deviation (SD)) of triplicates are presented.

IHC, hematoxylin-eosin (H&E) staining, and immunofluorescence analysis
IHC analysis was performed using an IHC kit (Cat.IHC001, Bioss, Beijing, China).Briefly, after deparaffinization and rehydration, the slides were boiled in 0.01 M, pH 8.0 ethylenediaminetetraacetic acid for antigen retrieval.Subsequently, the slides were incubated in 3% H 2 O 2 for 30 min to block endogenous peroxidase activity.
Next, the slides were incubated with primary antibodies for 2 h at 37 °C, followed by treatment with a secondary antibody and diaminobenzidine.Published standards were referred to establish cutoffs for "low" and "high" ZSWIM4 expression and Ki67 scores [24].For H&E staining, the sections were stained in hematoxylin for 5 min and eosin for 5 min.For the immunofluorescence assay, cells were fixed, permeabilized, and blocked, followed by incubation with primary antibodies overnight at 4 °C.Cells were then washed, incubated with secondary antibodies, and stained with DAPI (Cat.D-9106, Bioss).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay
AG RNAex Pro RNA reagent (Cat.AG21101), Evo M-MLV RT Kit with gDNA Clean for qPCR (Cat.AG11705), and SYBR Green Premix Pro Taq HS qPCR (Cat.AG11701) were obtained from Accurate Biotechnology (ChangSha, China).RNA extraction and cDNA synthesis were performed following the manufacturer's protocol.The resulting cDNA was then amplified on the Biosystems 7500 Real-time PCR system.The primers are listed in Additional file 1: Table S2.

Flow cytometry
The Annexin V-FITC reagent (Cat.KGF001, KeyGen) and the Annexin V-APC apoptosis detection kit (Cat.KGA1021, KeyGen) were used to detect apoptosis.A reactive oxygen species (ROS) detection kit (Cat.S0033S, Beyotime, Shanghai, China) was used to evaluate ROS levels according to the manufacturer's protocol.The anti-ZSWIM4/Cy3 antibody was used to detect ZSWIM4 protein expression levels in ZSWIM4-knockdown clones.All flow cytometry results were analyzed using the FlowJo software (Tree Star Inc., USA).

Tumorsphere assay
Briefly, 1 × 10 4 EOC cells were resuspended and plated on the ultra-low attachment six-well plates.The tumorspheres were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F-12 (Gibco) with epidermal growth factor, insulin, hydrocortisone, and B27 supplement.Cells were treated with 50 μM CBP for 5 days, and tumorspheres with a diameter > 100 μm were counted.

RNA-sequencing (RNA-seq) data analysis
Total RNA was isolated from ZSWIM4-knockdown and vector control OVCAR8 cells.A minimum of 5 μg total RNA was used for RNA-seq.RNA-seq and raw data analysis were performed by Novogene (Beijing, China).Gene Set Enrichment Analysis (GSEA) was performed for gene functional annotation and pathway analysis and the detailed results are presented in Additional file 1: Table S3.

Luciferase reporter assay
Briefly, cells were transfected with the reporter plasmids using Lipofectamine ™ 3000 (Cat.L3000015, Thermo Fisher Scientific).Luciferase activity was determined using a Microplate Reader (BioTek, Vermont, USA).ZSWIM4 gene luciferase reporter activities were normalized to that of the renilla luciferase.

CUT&Tag
For the CUT&Tag assay, a CUT&Tag-IT ™ Assay Kit, Anti-Rabbit (Cat.53,160, Active Motif, Shanghai, China) was used following the manufacturer's instructions.Briefly, OVCAR8 and SKOV3 cells were harvested and washed with pre-chilled PBS.FOXK1 antibody mixture was added to the cell lysate and incubated for 2 h at 25 °C.Subsequently, the cell lysate was further processed for 30 min incubation with a secondary antibody.After centrifugation, the Dig Buffer and CUT&Tag-Tn5 enzyme mixture was added and incubated at 25 °C for 1 h.Next, the products were purified, and the PCR cycle number was determined.To perform CUT&Tag sequencing, the Illumina NovaSeq 6000 sequencing platform was employed in the PE150 sequencing mode.The recommended sequencing volume gene was 6G, and additional sequencing volume was performed according to the actual requirements.CUT&Tag sequencing data processing was performed by 10 K Genomics, Shanghai, China.The IGV tool was used to visualize the read count data.

Chromatin immunoprecipitation (ChIP)
Approximately 5 × 10 6 OVCAR8 and SKOV3 cells were fixed with cross-link solution and collected.ChIP assays were performed using a ChIP Kit (Cat.abs50034, Absin, Shanghai, China) according to the manufacturer's instructions.Antibody-immunoprecipitated DNA was analyzed via real time PCR and normalized to the input.

Assessment of intracellular glycine content
A Glycine Assay Kit (Fluorometric; Cat.ab211100, Abcam) was used to detect cellular glycine concentrations.Briefly, the collected cells were counted and further lysed.After being deproteinized using 10 kDa spin columns, the filtrate was collected to determine intracellular glycine content.

Surface plasmon resonance (SPR) analysis
SPR analysis was used to detect the binding between the recombinant ZSWIM4 protein (Cat.TSTG2502, Tar-getMol) and IPN60090, with the ZSWIM4 protein and anti-ZSWIM4 antibody combination used as the positive control.The anti-ZSWIM4 antibody and IPN60090 were diluted to different concentrations using the running buffer (5% DMSO).The ZSWIM4 protein was coupled to the CM5 sensor chip (GE Healthcare, Marlborough, MA, USA) and served as the ligand.SPR experiments were performed using a BIAcore T200 instrument (GE Healthcare) at 25 °C following the manufacturer's instruction.The BIAcore T200 Control software (v.2.0, GE Healthcare) was used to analyze the binding model.

Bioinformatics analysis
Differential expression analysis for ZSWIM4 in ovarian tumors and control samples was performed using the Gene Expression Profiling Interactive Analysis (GEPIA) database [25].The survival analysis based on ZSWIM4 expression in OC was implemented using the GEPIA database and Kaplan-Meier plotter [26].The gene expression levels of ZSWIM4 and FOXK1 in The Cancer Genome Atlas database (TCGA) and Etemadmoghadam 2009 were obtained from the UCSC Xena project [27], and the expression of ZSWIM4 and FOXK1 in platinumresistant OVCAR8 cells were obtained from GSE45553 [28].Virtual screening of ZSWIM4 targeted inhibitors was performed using Schrödinger Maestro ver.11.4, and molecular docking between ZSWIM4 and IPN60090 was conducted using PyMol.

Patient-derived organoid (PDO) model construction and culture
Fresh tumor tissues obtained from EOC patients were immersed in Advanced DMEM/F12 (Cat.12634028, Thermo Fisher Scientific) containing 1% penicillinstreptomycin (PS).The necrotic tissues were removed, and the remaining tissue was cut into pieces of approximately 1-2 mm 3 volume.After washing twice with PBS containing 1% PS, the tissue fragments were digested at 37 °C for 1 h using the Advanced DMEM/ F12 solution containing type IV Collagenase (Cat.17104019, Thermo Fisher Scientific).The above solution was filtered using a 100 μm filter (Cat.352360, Corning, New York, USA).Next, the fragments were centrifuged at 300×g for 5 min.The erythrocytes were lysed.The precipitated cells were resuspended in the Advanced DMEM/F12 mixed with growth factorreduced Matrigel (Cat.CB-40230, Corning, USA).The cell suspension was adjusted to a final concentration of 10,000-20,000 cells/50 μl.PDOs were transferred to 24-well plates when the Matrigel solidified, and 500 μl of the general culture medium was added.PDOs were grown in a cell incubator at 37 °C with 5% CO 2 .The culture medium was changed every 2-3 days.The cell viability of PDOs was determined by the Cell Counting-Lite 3D Luminescent Cell Viability Assay (Cat.DD1102-01, Vazyme, Nanjing, China).

Statistical analysis
Excluding the ZSWIM4 IHC results, which were expressed as median with interquartile range, all other data were expressed as mean ± SD and were analyzed using GraphPad Prism (6.0) software.The two groups were compared to obtain significant differences using Student's t-test (two-tailed) or Welch's test for data with unequal variance.A one-way analysis of variance (ANOVA) was used to compare multiple groups, and a Mann-Whitney U-test was performed for IHC score data.A two-way ANOVA was performed to assess significant differences in tumor volume growth curves.The Fisher's exact test was adopted to evaluate differences in relative ZSWIM4 expression.The Gehan-Reslow-Wilcoxon test was used to determine the significance of survival data.Pearson's correlation was used to determine the correlation between ZSWIM4 and FOXK1 expression.P values < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001) were considered significant, otherwise non-significant (ns).

ZSWIM4 is highly expressed in EOC and is an indicator of poor prognosis in patients
First, by analyzing the data from the GEPIA database, we found that ZSWIM4 was overexpressed in OC tumor tissues (Additional file 1: Fig. S1A), and that poor prognosis was associated with high ZSWIM4 expression (Additional file 1: Fig. S1B).We consistently confirmed poorer prognosis in patients with OC showing ZSWIM4 overexpression using the Kaplan-Meier plotter website (Additional file 1: Fig. S1C, D).Subsequently, we performed IHC to verify the overexpression of ZSWIM4 protein using EOC tumor tissues (n = 15) and control tissue samples (n = 15), including 10 paired samples.Interestingly, 11 of the 15 cancer tissues showed high ZSWIM4 expression, whereas only four of the 15 normal control tissues showed ZSWIM4 expression, indicating the upregulation of ZSWIM4 protein levels in EOC (Fig. 1A, B).Subsequently, we performed an IHC analysis for ZSWIM4 using an OC TMA.Based on the staining intensity and pattern, 114 valid ZSWIM4 expression scores were collected.In those 114 EOC tumors, the expression of ZSWIM4 among different EOC tissue types showed no significant difference (Additional file 1: Fig. S1E).Notably, we found a significant increase of ZSWIM4 expression in relapsed primary cancers (Fig. 1C, D) and a positive correlation between high ZSWIM4 expression and primary tumor recurrence based on the clinical information (Fig. 1E).Survival analysis results based on the TMA also revealed that high expression of ZSWIM4 could predict poorer overall survival rates (Fig. 1F) and higher postoperative relapse rates (Fig. 1G) in patients with EOC.Furthermore, to investigate the factors contributing to the effects of ZSWIM4 on EOC recurrence, we evaluated its prognostic role in patients with this disease treated with platinum-based chemotherapy using the Kaplan-Meier plotter database.Notably, high ZSWIM4 expression in OC indicated a poor prognosis for the patients administered platinumbased treatment (Fig. 1H), suggesting that high ZSWIM4 expression in EOC tumor tissues might counter the effect of platinum drugs.

Elevated ZSWIM4 expression reduces EOC cell sensitivity to CBP
To investigate the effect of ZSWIM4 in driving chemotherapy resistance, we selected two cell lines with low expression of this protein, IOSE-80 (human normal ovarian epithelial cells) and ES-2 (human ovarian clear cell carcinoma cells) cells (Additional file 1: Fig. S2A), to establish the ZSWIM4 overexpression models (Fig. 2A, Additional file 1: Fig. S2B).Subsequently, we treated both vector control and ZSWIM4-overexpressing clones with 50 μM CBP for 72 h, and observed that overexpression of ZSWIM4 conferred resistance to CBP in IOSE-80 and ES-2 cell lines (Additional file 1: Fig. S2C).In addition, exogenous ZSWIM4 expression increased the IC 50 values of CBP in both IOSE-80 and ES-2 cell lines (Fig. 2B).In our previous study, we found that ZSWIM4 was an inducible drug resistance gene in breast cancer cells [14].Notably, the messenger RNA (mRNA) (Additional file 1: Fig. S2D) and protein expression levels (Fig. 2C) of ZSWIM4 were significantly induced upon 75 μM CBP treatment in OVCAR8 and SKOV3 cells.To discern the pathological significance of inducible ZSWIM4 in EOC cells upon CBP treatment, stable clones of ZSWIM4-overexpressing OVCAR8 and SKOV3 cells were generated to mimic the above situations (Fig. 2D, Additional file 1: Fig. S2E).Then, both vector control and ZSWIM4-overexpressing EOC cells were incubated with 100 μM CBP for 72 h.We found that the up-regulation of ZSWIM4 compromised the cytotoxic effect of CBP, as indicated by the CCK-8 assay results (Additional file 1: Fig. S2F).Furthermore, we generated IC 50 curves and found that ZSWIM4 overexpression increased the IC 50 values of CBP (Fig. 2E).Moreover, the overexpression of ZSWIM4 weakened CBP-mediated suppression of tumorsphere formation in OVCAR8 and SKOV3 cells (Additional file 1: Fig. S2G).Subsequently, we characterized CBP-induced cell apoptosis in vector control and ZSWIM4-overexpressing OVCAR8 and SKOV3 cells incubated with 50 μM CBP for 72 h.Overexpression of ZSWIM4 significantly ameliorated CBP-induced apoptosis, as evidenced by the reduced Annexin V-positive cell proportions (Fig. 2F, Additional file 1: Fig. S2H) and the downregulation of cleaved caspase-3 (Fig. 2G).These data suggest that high ZSWIM4 expression in EOC cells could reduce their sensitivity to CBP by inhibiting CBPmediated apoptosis.

Silencing ZSWIM4 enhances EOC cell sensitivity to CBP
To better clarify the impact of ZSWIM4 on chemosensitivity, we knocked down ZSWIM4 in OVCAR8 and SKOV3 cells (Fig. 3A, Additional file 1: Fig. S3A).Subsequently, we treated ZSWIM4-knockdown OVCAR8 cells and control cells with 100 μM CBP and ZSWIM4-knockdown SKOV3 cells and control cells with 50 μM CBP for 72 h.Cell viability assays unveiled that ZSWIM4-knockdown EOC cells were more sensitive to CBP treatment (Fig. 3B).Furthermore, after incubation with a gradient concentration of CBP for 72 h, the IC 50 values declined in both ZSWIM4-knockdown EOC cells (Fig. 3C), indicating that ZSWIM4 knockdown enhances EOC cell sensitivity to chemotherapy.ZSWIM4 silencing facilitated the CBP-mediated inhibition of tumorsphere formation (Fig. 3D, Additional file 1: Fig. S3B) and enhanced CBP-mediated apoptosis (Fig. 3E, Additional file 1: Fig. S3C).Consistently, the increased expression of cleaved caspase-3 in the CBP-treated ZSWIM4-knockdown EOC clones validated the above results (Fig. 3F).Furthermore, we analyzed the response of vector control cells and ZSWIM4-knockdown cells to CBP in vivo.For this, we injected the ZSWIM4-knockdown or control SKOV3 cells into nude mice.Then, CBP treatment at a dose of 20 mg/kg three times per week was initiated when xenograft tumor volume reached approximately  3 .After 2 weeks of treatment, we found that the knockdown of ZSWIM4 also sensitized EOC cells to CBP in vivo (Fig. 3G), as indicated by the slower growth curve (Fig. 3H) and reduced tumor weights (Fig. 3I) of subcutaneous tumors among the four groups of mice.The decreased Ki67 proliferation score also confirmed these results (Additional file 1: Fig. S3D).These data

FOXK1-dependent ZSWIM4 transcription reduces EOC cell sensitivity to CBP
We explored the mechanism underlying the induction of ZSWIM4 expression upon CBP treatment.A previous study showed that the transcription factor FOXK1 is upregulated in platinum drug-resistant cell lines [29].Based on public database analysis, strong positive correlations between FOXK1 and ZSWIM4 mRNA levels were observed in OC tumor tissues (Additional file 1: Fig. S4A).By analyzing gene expression in OVCAR8 parental cells and cisplatin-resistant cells, we observed co-upregulation of FOXK1 and ZSWIM4 in cisplatin-resistant OVCAR8 cells (Additional file 1: Fig. S4B).Our western blotting results revealed that FOXK1 abundance gradually increased upon CBP (75 μM) treatment within 72 h (Fig. 4A).By searching the promoter region of ZSWIM4 in the JASPAR database, setting a filtering criterion of a score greater than 10 and a correlation greater than 0.9, we identified two potential FOXK1-binding sites (Additional file 1: Fig. S4C).Next, we performed CUT&Tagseq using an antibody against FOXK1 (Additional file 1: Fig. S4D) and found the common binding peak of FOXK1 in the promoter region of ZSWIM4 loci in both OVCAR8 and SKOV3 cells (Fig. 4B).The ChIP assays were then conducted, and the results confirmed that FOXK1 bound directly to the ZSWIM4 promoter region (Fig. 4C, D).Therefore, we hypothesized that ZSWIM4 upregulation might be mediated by the transcriptional effect of FOXK1.To test our hypothesis, we constructed a wild-type ZSWIM4 promoter-luciferase reporter vector by inserting the 1500 bp promoter fragment upstream of the ZSWIM4 gene translation start site and three mutated ZSWIM4 promoter-luciferase reporter vectors in which the FOXK1-binding sites were mutated (MUT #1, MUT #2, and MUT #3) (Additional file 1: Fig. S4E).As expected, 100 μM CBP treatment for 24 h enhanced ZSWIM4 gene transcriptional activity in both OVCAR8 and SKOV3 cells, whereas the mutation in the FOXK1-recognition motif eliminated CBP-induced luciferase activity.Notably, mutations in two binding sites had a more significant effect on reducing the CBPinduced upregulation of ZSWIM4 transcriptional activity than mutations in any single binding site (Fig. 4E).Furthermore, we knocked down FOXK1 in OVCAR8 and SKOV3 cells to elucidate its role in inducing ZSWIM4 (Fig. 4F).Notably, the knockdown of FOXK1 downregulated ZSWIM4 (Additional file 1: Fig. S4F) and its transcriptional activity (Additional file 1: Fig. S4G).Simultaneously, silencing FOXK1 reduced the upregulation of ZSWIM4 promoter-luciferase activity (Fig. 4G) and inhibited the upregulation of ZSWIM4 induced by 75 μM CBP treatment for 48 h (Fig. 4H).We also generated stable FOXK1-overexpressing clones in OVCAR8 cells (Additional file 1: Fig. S4H).In line with our hypothesis, ectopic FOXK1 expression enhanced CBP-induced ZSWIM4 promoter-luciferase activity (Fig. 4I).Moreover, knockdown of ZSWIM4 in FOXK1-overexpressing OVCAR8 cells partially alleviated drug resistance to CBP (Additional file 1: Fig. S4I).Collectively, our results indicated that FOXK1-mediated ZSWIM4 transcription was responsible for the decreased sensitivity of EOC cells to CBP.

ZSWIM4 knockdown enhances CBP-induced reactive oxygen species (ROS) production
To elucidate how ZSWIM4 confers decreased sensitivity to CBP in EOC cells, we performed next-generation RNA-seq using ZSWIM4-knockdown and control OVCAR8 cells.Pathway enrichment analysis demonstrated that "GLYCINE_SERINE_AND_THREO-NINE_METABOLISM" was the most significantly downregulated pathway after ZSWIM4 knockdown (Fig. 5A, Additional file 1: Fig. S5A).By investigating the members of this pathway, we found that most of the downregulated genes-encoded key enzymes participate in the synthesis of serine and glycine (Additional file 1: Fig. S5B).Among them, the expression levels of PHGDH, the gene encoding phosphoglycerate dehydrogenase, and SHMT2, the gene encoding serine hydroxymethyltransferase 2, were the lowest (Fig. 5B).In addition, we confirmed that ZSWIM4 knockdown reduced the expression of PHGDH and SHMT2 (Fig. 5C), whereas the overexpression of ZSWIM4 upregulated PHGDH and SHMT2 expression in OVCAR8 and SKOV3 cells (Fig. 5D).PHGDH catalyzes intracellular serine biosynthesis [30].Serine is further converted into glycine by SHMT, and SHMT2 is the rate-limiting enzyme in this step [31].A previous study indicated that PHGDH knockdown enhances EOC cell chemosensitivity [32].Notably, in both EOC cell lines, ZSWIM4 knockdown decreased the intracellular glycine content (Fig. 5E), whereas exogenous glycine supplementation partially restored the sensitivity of OVCAR8 cells to CBP induced by ZSWIM4 knockdown (Additional file 1: Fig. S5C).Therefore, we investigated whether the downregulation of SHMT2 after ZSWIM4 knockdown could also enhance the chemotherapy sensitivity in EOC cells.For this, we knocked down SHMT2 in OVCAR8 and SKOV3 cells and observed that silencing SHMT2 enhanced the sensitivity of CBP treatment in both two EOC cell lines (Additional file 1: Fig. S5D).Consistently, 50 or 100 µM CBP in combination with the SHMT inhibitor (SHIN1) showed a stronger killing effect in both OVCAR8 and SKOV3 cells than CBP treatment alone  (Additional file 1: Fig. S5E).Next, we knocked down SHMT2 in ZSWIM4-overexpressing clones and treated control and SHMT2-knockdown cells with 100 µM CBP.SHMT2 silencing partially rescued the reduced sensitivity of CBP caused by ZSWIM4 overexpression in both EOC cell lines (Fig. 5F), indicating that SHMT2 is a downstream target of ZSWIM4.SHMT2 catalyzes the synthesis of glycine, which is involved in various metabolic pathways, such as purine nucleotide and GSH synthesis [33].We treated control cells and ZSWIM4-knockdown OVCAR8 cells with 100 µM CBP and exogenously administered hypoxanthine (100 µM) and GSH (100 µM) to the knockdown group.Interestingly, GSH partially rescued the effects of ZSWIM4 knockdown, whereas hypoxanthine did not (Fig. 5G).GSH is an important regulator of intracellular redox homeostasis [34].Furthermore, we observed a CBP-induced increase in intracellular ROS levels following ZSWIM4 knockdown (Fig. 5H) and decreased ROS levels in ZSWIM4-overexpressing OVCAR8 cells after CBP (50 μM) treatment (Fig. 5I).These results suggest that ZSWIM4 knockdown downregulates the glycine synthesis enzymes, leading to a diminution in intracellular glycine content, and augments the CBP-induced ROS levels, thereby enhancing the effect of CBP on EOC cells.

A ZSWIM4 inhibitor enhances the sensitivity of EOC cells to CBP
Currently, there are few reports on ZSWIM4-targeting inhibitors.Therefore, we attempted to identify small-molecule inhibitors targeting this protein using computerized virtual screening.We predicted the threedimensional (3D) structure of ZSWIM4 using Alpha-Fold, and a high percentage of 3D structures scored > 70 points (Fig. 6A), indicating that this prediction had high structural reliability.Next, we found that the Site1 binding pocket in the 3D structure had a strong ability to accept compounds (Additional file 1: Fig. S6A).Therefore, we performed a virtual screening against this Site1 locus using the MedChemExpress Bioactive Compound Library Plus and yielded three small-molecule compounds (mirabegron, IPN60090, and nelociguat) with a docking score of less than ˗10.5 (Additional file 1: Fig. S6B).Subsequently, we treated OVCAR8 cells with 25 μM CBP in combination with the three drugs (all at a concentration of 10 μM).Notably, only IPN60090 enhanced the sensitivity of OVCAR8 cells to CBP (Additional file 1: Fig. S6C).IPN60090 was initially developed as an inhibitor of glutaminase-1 (GLS-1), and it has entered clinical trials for cancer therapy, especially for lung and ovarian cancers [35,36].Therefore, we further investigated the interaction between IPN60090 and ZSWIM4.Docking results indicated that IPN60090 (Additional file 1: Fig. S6D) binds to the ZSWIM4 protein by forming hydrogen bonds with the residues proline 204 (Pro372) and glycine 411 (Gly411) in ZSWIM4 (Fig. 6B).Subsequently, using an anti-ZSWIM4 antibody as positive control (Fig. 6C), we performed an SPR assay to detect the affinity between the recombinant ZSWIM4 protein (Additional file 1: Fig. S6E) and IPN60090.The results showed that IPN60090 can specifically bind to the ZSWIM4 protein (Fig. 6D).Furthermore, IPN60090 was found to downregulate the intracellular glycine levels (Fig. 6E) and elevate ROS levels (Fig. 6F, Additional file 1: Fig. S6F) in OVCAR8 and SKOV3 cells.Furthermore, cell viability assays showed that 50 μM CBP in combination with 10 μM IPN60090 exhibited a stronger cytotoxic effect (Additional file 1: Fig. S6G), and IC 50 curve analyses revealed that the ZSWIM4 inhibitor had a chemosensitizing effect (Additional file 1: Fig. 6G).In addition, the combination of IPN60090 and CBP inhibited the formation of tumorspheres in both OVCAR8 and SKOV3 cells (Fig. 6H, Fig. S6H).To clarify whether IPN60090 exerts CBP-sensitizing effects by inhibiting ZSWIM4, we treated both ZSWIM4-knockdown and control EOC cells with 25 μM CBP alone or 25 μM CBP combined with IPN60090 (10 μM) for 72 h.Interestingly, IPN60090 lost its synergistic effect with CBP in ZSWIM4-knockdown cells (Fig. 6I).Consistently, IC 50 curve analyses suggested that IPN60090 could be used to overcome CBP resistance caused by ZSWIM4 overexpression in OVCAR8 cells (Additional file 1: Fig. S6I).Considering that poly (ADP-ribose) polymerase (PARP) inhibitors are also used for the clinical treatment of EOC [1] and metabolic reprogramming can cause EOC cells to resist PARP inhibitors [21], we attempted to clarify whether the use of ZSWIM4 inhibitors could also enhance the sensitivity to PARP inhibitors.We treated both OVCAR8 and SKOV3 cells with 50 or 100 µM olaparib in combination with IPN60090 (50 µM) for 48 h, and the results showed that this cotreatment resulted in a stronger killing effect than olaparib treatment alone (Additional file 1: Fig. S6J).Furthermore, we evaluated the chemotherapeutic sensitization effect of the ZSWIM4 inhibitor IPN60090 in vivo using SKOV3-derived xenograft mice.When the tumor volume reached 100 mm 3 , the mice were administered the vehicle control, CBP (15 mg/kg, i.p.) 3 weekly, IPN60090 (30 mg/kg/day, per os (p.o.)), and CBP combined with IPN60090.After 10 days of drug treatment, the mice were euthanized.IPN60090 significantly enhanced the inhibitory effect of CBP in vivo (Fig. 6J-L).Collectively, our data revealed that the use of a ZSWIM4 inhibitor exerts marked synergistic effects with CBP in killing EOC cells, like those with ZSWIM4 knockdown.The ZSWIM4 inhibitor enhances CBP sensitivity in CBP-resistant EOC cells Furthermore, we established CBP-resistant SKOV3 clones (SKOV3/CBP) using parental SKOV3 cells by increasing the concentration of CBP stepwise.The IC 50 value of CBP in SKOV3/CBP cells was more than five-fold higher than that in the parental SKOV3 cells (Fig. 7A).We observed higher FOXK1 protein abundance in SKOV3/CBP cells than in the parental cells (Fig. 7B).ZSWIM4 was consistently upregulated in the CBPresistant clones (Fig. 7C).Thereafter, we transfected the ZSWIM4 promoter-luciferase plasmid into both parental and CBP-resistant SKOV3 cells and obtained an elevated luciferase reporter activity in SKOV3/CBP cells (Fig. 7D).These data suggested that FOXK1/ZSWIM4 signaling was highly activated in SKOV3/CBP cells.Subsequently, we treated SKOV3/CBP cells with 200 μM CBP combined with or without 50 μM IPN60090.As expected, the co-treatment resulted in a better killing effect (Additional file 1: Fig. S7A).Moreover, IC 50 curve analyses revealed that the addition of the ZSWIM4 inhibitor re-sensitized SKOV3/CBP cells to CBP (Fig. 7E).Flow cytometric results further confirmed that co-treatment increased intracellular ROS levels (Fig. 7F, G) and robustly enhanced apoptosis in SKOV3/CBP cells (Fig. 7H, Additional file 1: Fig. S7B).Western blotting results also revealed that CBP (200 μM) co-treated with IPN60090 (50 μM) induced markedly stronger expression of cleaved caspase-3 (Fig. 7I).Collectively, our data support the hypothesis that ZSWIM4 inhibition sensitizes EOC cells to chemotherapy.

ZSWIM4 inhibition sensitizes EOC cells to chemotherapy in PDO models
CBP in combination with paclitaxel (PTX) is the first-line regimen for the clinical treatment of EOC [2].We investigated whether the inhibition of ZSWIM4 could enhance the efficacy of this strategy.For this, we treated ZSWIM4knockdown and control OVCAR8 cells with CBP (25 μM) alone or in combination with PTX (5 nM).Cell viability assays indicated that ZSWIM4-knockdown also sensitized EOC cells to this chemotherapy (Additional file 1: Fig. S8A).In both OVCAR8 and SKOV3 cells, the triple dosing regimen of CBP, PTX, and IPN60090 had an optimal inhibitory effect on cell viability (Additional file 1: Fig. S8B), and the use of ZSWIM4 inhibitors enhanced apoptosis induced by the combination of CBP and PTX (Additional file 1: Fig. S8C, D).PDO models are important preclinical models for evaluating drug responses and efficacy.To explore the potential clinical use of ZSWIM4-targeted inhibitors, tumor tissues exhibiting ZSWIM4 expression from two patients with EOC were obtained to construct PDO models (Fig. 8A).We then performed combination drug experiments using these two PDO models with the following treatments: vehicle, CBP (100 μM) combined with PTX (10 nM), IPN60090 (50 μM), and CBP combined with PTX plus IPN60090.Cell viability of the PDO models was measured on days four and seven using an adenosine triphosphate (ATP) detection kit.Notably, IPN60090 enhanced EOC cell sensitivity to chemotherapy in PDOs expressing ZSWIM4 (Fig. 8B, C).Collectively, these data suggest that therapeutic strategies targeting ZSWIM4 in patients with high expression of this marker may enhance the sensitivity of EOC cells to chemotherapy.

Discussion
Chemotherapy resistance challenges the clinical treatment of EOC patients, and more than 60% of the patients are prone to recurrence and resistance to first-line chemotherapy drugs [37].The development of chemoresistance in tumor cells is typically caused by abnormal transcriptional regulation patterns [38].FOXK1 is an important transcription factor of aerobic glycolysis [39], which is closely associated with tumorigenesis, including triple negative breast cancer [40], gallbladder cancer [41], hepatocellular carcinoma [42], and ovarian malignancy [43].A recent study reported that the Aurora-A/sexdetermining region Y box-containing transcription factor 8 (SOX8)/FOXK1 axis enhances chemotherapy resistance in EOC cells and that inhibition of the transcription function of FOXK1 improves the therapeutic efficacy in platinum-resistant cells [29].Hence, abnormal activation of FOXK1 facilitates the development or chemoresistance of EOC and could be regarded as a candidate therapeutic target against EOC.Indeed, clarifying the role of FOXK1 in EOC drug resistance will provide more favorable conditions for reducing chemotherapy resistance.Our study provided a novel mechanism of chemoresistance in which EOC cells survive in the presence of CBP by inducing ZSWIM4 expression via FOXK1-mediated transcription.However, the transcriptional activity of ZSWIM4 was not fully lost after FOXK1 knockdown, indicating that high expression of ZSWIM4 in EOC was also regulated by other factors.Therefore, the molecular mechanisms underlying the upregulation of ZSWIM4 in EOC tumor tissues require further investigation.
The ZSWIM family proteins can interact with DNA or proteins and exert powerful regulatory effects on cells because of their unique SWIM domain [8].Several studies have shown that ZSWIM5 is important in regulating neuronal development [44].Recent studies have also reported that ZSWIM8 participates in protein ubiquitination as a ubiquitin ligase [10].However, specific biological functions of ZSWIM4 have rarely been reported.In this study, through public database analysis and clinical sample validation, we observed a positive correlation between high ZSWIM4 expression and EOC recurrence and a restricted efficacy of platinum-based chemotherapy in patients with ZSWIM4 overexpression.In addition, ZSWIM4 inhibition exerts a significant chemo-sensitization effect in vivo and in vitro, as well as in PDO models, indicating that ZSWIM4 may serve as a therapeutic target and molecular marker for the prognosis of patients with EOC.By integrating bulk RNA-seq, signaling pathway analysis, and molecular and biochemical approaches, we found that ZSWIM4 knockdown impacts glycine biosynthesis, increasing cellular ROS levels in EOC cells and sensitizing them to chemotherapeutic drugs.However, the mechanism through which ZSWIM4 regulates the expression of these key enzymes remains obscure.Notably, ZSWIM4 inhibition showed little impact on EOC cell proliferation, whereas the inhibition of glycine affected the growth of tumor cells, indicating that there is a compensatory mechanism in ZSWIM4-knockdown EOC cells to overcome this damage.Hence, a compensatory model associated with combined inhibition might have a killing effect on cells with high ZSWIM4 expression.Furthermore, we found that knockdown of ZSWIM4 impacts the pentose phosphate pathway in EOC cells, further demonstrating the importance of ZSWIM4 in regulating EOC cell metabolism.Hence, further exploration of potential biological functions of ZSWIM4 in EOC will be carried out in the future.Considering the lack of targeted inhibitors for ZSWIM4, we sought to identify small molecules from existing compound libraries that bind to, and potentially inhibit, ZSWIM4.The small-molecule compound IPN60090 is an orally active inhibitor of GLS-1, with no activity observed against GLS-2 [35].GLS-1 inhibition alone or in combination with immune checkpoint blockade represents an effective therapeutic strategy for SWI/ SNF-altered cancers such as ARID1A-mutated ovarian clear cell carcinoma [45,46].Here, we found that IPN60090 binds to ZSWIM4 and simulates the chemosensitization effect caused by ZSWIM4 knockdown.We will further elucidate how IPN60090 can be used to overcome chemotherapy resistance in EOC cells by altering intracellular metabolic processes in the following study.Subsequently, we also observed the activation of the FOXK1/ZSWIM4 axis and a significant synergistic effect between IPN60090 and CBP in SKOV3/CBP cells, suggesting that the resistance of SKOV3/CBP against CBP depends on the expression of ZSWIM4.Our study provides a compound framework for developing ZSWIM4 inhibitors.However, the ZSWIM4 inhibitor did not fully reverse the IC 50 values of EOC cells to CBP, indicating the complexity of chemoresistance in EOC cells, which requires further investigation.
Due to the heterogeneity of EOC cells, the treatment responses and outcomes vary.Unfortunately, we are unable to fully evaluate the expression of ZSWIM4 in different pathological types of EOC.This implies that the TMA results can only suggest that ZSWIM4 expression may be higher in serous carcinoma than in clear cell carcinoma.Thus, to accurately predict the prognosis, we will expand the EOC sample set to evaluate ZSWIM4 protein expression in different EOC cell types.Overall, our study demonstrates the aberrant expression of ZSWIM4 in EOC and its involvement in chemosensitivity, suggesting that targeting ZSWIM4 could overcome EOC chemotherapy resistance.

Conclusions
ZSWIM4 was overexpressed in EOC tumor tissues and was relevant to a poor prognosis.Furthermore, ZSWIM4 silencing promoted CBP-induced apoptosis and enhanced chemotherapy sensitivity in EOC cells.Moreover, ZSWIM4 was upregulated by FOXK1 following CBP treatment in EOC cells.Functionally, ZSWIM4 regulated the intracellular glycine metabolism to induce chemoresistance, and the ZSWIM4 inhibitor, IPN60090, sensitized EOC cells to chemotherapy by increasing cellular ROS levels induced by CBP (Fig. 9).These findings

Fig. 1
Fig. 1 ZSWIM4 is highly expressed in epithelial ovarian cancer (EOC) and indicates poor prognosis.A IHC staining for ZSWIM4 in EOC tumor tissues and adjacent tissues; representative graphs are presented.B Statistical analysis of ZSWIM4 IHC score in EOC tumor tissues (n = 15) and adjacent tissues (n = 15).C IHC staining of ZSWIM4 in primary tumor tissues from patients with recurrent and non-recurrent EOC; representative graphs are presented.D IHC scores of ZSWIM4 in the recurrent and non-recurrent groups.Re, recurrence.E Statistical analysis of ZSWIM4 protein expression in primary tumor tissues from patients with recurrent (n = 91) and non-recurrent (n = 23) EOC.Re, recurrence.F, G Survival analysis of 114 EOC patients based on ZSWIM4 expression in TMA.Overall survival F. Relapse-free survival G. H Survival analysis of ZSWIM4 expression in OC patients treated with platinum-based drugs using the Kaplan-Meier plotter website

Fig. 2
Fig. 2 Elevated ZSWIM4 resists chemotherapy in epithelial ovarian cancer (EOC) cells.A ZSWIM4 protein expression levels in the vector control and ZSWIM4-overexpressing IOSE-80 and ES-2 cells.B IC 50 curves for vector control and ZSWIM4-overexpressing IOSE-80 and ES-2 cells following incubation with a gradient concentration CBP for 72 h.C Immunofluorescence detection of ZSWIM4 protein expression in OVCAR8 and SKOV3 cells after CBP (75 μM) treatment for 48 or 72 h.D ZSWIM4 protein expression in the vector control and ZSWIM4-overexpressing OVCAR8 and SKOV3 cells.E IC 50 curves for vector control and ZSWIM4-overexpressing OVCAR8 and SKOV3 cells following incubation with CBP for 72 h.F Statistical data (mean ± SD) of the apoptosis rate in ZSWIM4-overexpressing and vector control OVCAR8 and SKOV3 cells treated with the vehicle control or CBP (50 μM) treatment for 72 h.G Cleaved caspase-3 protein levels in ZSWIM4-overexpressing OVCAR8 and SKOV3 cells treated with CBP (50 μM) for 72 h detected via western blotting

Fig. 3
Fig. 3 Silencing ZSWIM4 enhances the sensitivity of epithelial ovarian cancer (EOC) cells to CBP.A ZSWIM4 protein expression levels in the vector control and shRNA-mediated ZSWIM4-knockdown EOC clones.Representative flow cytometry histograms (left) and statistical data (mean ± SD) are shown (right).B Killing effects in ZSWIM4-knockdown and shRNA control OVCAR8 cells treated with CBP (100 μM) for 72 h and ZSWIM4-knockdown and shRNA control SKOV3 cells treated with CBP (50 μM) for 72 h; CCK-8 assays.C CBP sensitivity of ZSWIM4-knockdown OVCAR8 and SKOV3 cells.D The statistical data (mean ± SD) of tumorspheres of control and ZSWIM4-knockdown EOC cells incubated in 50 μM CBP for 5 days.E The statistical data (mean ± SD) of apoptosis rate of ZSWIM4-knockdown and control EOC cells with or without CBP (50 μM) treatment for 72 h.F The protein expression levels of cleaved caspase-3 in CBP-treated ZSWIM4-knockdown EOC cells.shC, shCTR.G-I The shRNA control and ZSWIM4-knockdown SKOV3-derived xenograft mice were treated with vehicle control or CBP (20 mg/kg thrice weekly) for two weeks (n = 8/group).Photographs of tumors G. Growth curves of xenograft tumors H. Tumor weight I. Veh, vehicle

Fig. 4
Fig. 4 FOXK1-dependent ZSWIM4 transcription contributes to epithelial ovarian cancer (EOC) cell chemotherapy resistance.A FOXK1 protein levels in both EOC cell lines treated with vehicle or CBP (75 μM) for 0, 48, and 72 h.B CUT&Tag-seq analyses of FOXK1 binding peaks on the promoter regions of ZSWIM4 in OVCAR8 and SKOV3 cells.Rep-1, replicated sample-1, Rep-2, replicated sample-2.C Enrichment of ZSWIM4 promoter fragments using an antibody against FOXK1 was assessed by ChIP-qPCR analysis.D Agarose electrophoresis analysis of PCR products of the ZSWIM4 promoter after ChIP-PCR assays.E ZSWIM4 promoter activity in EOC cells transfected with wild-type or mutant plasmids in the presence or absence of 100 μM CBP for 24 h.F FOXK1 knocked down in OVCAR8 and SKOV3 cells.G ZSWIM4 promoter activity in FOXK1 silencing EOC cells treated with vehicle or CBP (75 μM) for 48 h.H Immunofluorescence analysis of ZSWIM4 expression in control and FOXK1-knockdown OVCAR8 cells treated with CBP (75 μM) for 48 h.I ZSWIM4 promoter activity in OVCAR8 cells transfected with wild-type ZSWIM4 promoter-luciferase plasmid with or without FOXK1 co-transfection.The cells were treated with vehicle or CBP (75 μM) for 48 h

Fig. 5
Fig. 5 Glycine metabolism reprogramming is triggered after knocking down ZSWIM4 in EOC cells.A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in OVCAR8 cells after ZSWIM4 knockdown.B The mRNA expression levels of genes encoding key enzymes for glycine synthesis in vector control and ZSWIM4-knockdown OVCAR8 cells.C, D Protein expression levels of PHGDH and SHMT2 in ZSWIM4-knockdown EOC clones C and ZSWIM4-overexpressing EOC clones D. E Intracellular glycine content of ZSWIM4-knockdown OVCAR8 and SKOV3 cells.F Viability of cells following SHMT2 knockdown in vector control and ZSWIM4-overexpressing EOC cells treated with vehicle or CBP (100 μM) for 72 h; CCK-8 assay.G Rescue experiments using exogenous supplementation of hypoxanthine and GSH in ZSWIM4-knockdown OVCAR8 cells.H ROS levels in vector control and ZSWIM4-knockdown OVCAR8 cells after CBP (50 μM) treatment for 72 h.Representative histograms (upper) and statistical data (mean ± SD) are shown (lower).I Flow cytometry detection of ROS levels in vector control and ZSWIM4-overexpressing OVCAR8 cells treated with vehicle or CBP (50 μM) for 72 h.Representative histograms (upper) and statistical data (mean ± SD) (lower) are shown

Fig. 6
Fig. 6 IPN60090, a ZSWIM4-targeting inhibitor, enhances the chemotherapeutic sensitivity of EOC cells.A 3D structure of human ZSWIM4.B Structural simulation of ZSWIM4 and IPN60090.IPN60090 is depicted as a gray-white stick, with the red dashed line indicating the length of the hydrogen bond.C-D The affinity of anti-ZSWIM4 antibody C and IPN60090 D for ZSWIM4 protein determined by SPR analyses.KD, dissociation constant.E Detection of intracellular glycine content in OVCAR8 and SKOV3 cells treated with 10 μM IPN60090 for 48 h.F ROS levels in EOC cells following IPN60090 (10 μM) treatment for 48 h.G IC 50 curves for OVCAR8 and SKOV3 cells following treatment with CBP and/ or IPN60090 (10 μM) for 72 h.H Tumorsphere formation in OVCAR8 cells treated with vehicle control, CBP (50 μM), IPN60090 (10 μM), and CBP (50 μM) combined with IPN60090 (10 μM) for 5 day.Representative images (left) and statistical data (mean ± SD) (right) are shown.I Viability of ZSWIM4-knockdown and vector control EOC cells following treatment with CBP (25 μM) with or without IPN60090 (10 μM) for 72 h; CCK-8 assay.J-L SKOV3-derived xenograft mice were divided into four groups (n = 4/group) and administered the vehicle control, CBP (15 mg/kg, i.p.) thrice weekly, IPN60090 (30 mg/kg/day, p.o.), and CBP combined with IPN60090.Photographs of tumors J. Tumor weight K. Growth curves of xenograft tumors L

Fig. 7
Fig. 7 ZSWIM4 inhibitor enhances chemotherapy sensitivity in CBP-resistant SKOV3 cells.A Drug sensitivity of CBP in parental SKOV3 and SKOV3/ CBP cells.B The protein expression of FOXK1 in parental SKOV3 and SKOV3/CBP cells.C Immunofluorescence detection of ZSWIM4 protein expression in parental SKOV3 and SKOV3/CBP cells.D ZSWIM4 gene promoter-luciferase reporter activity in parental SKOV3 and SKOV3/CBP cells.E IC 50 curves of SKOV3/CBP cells with a gradient concentration of CBP with or without IPN60090 (50 μM) treatment for 72 h.(F-I) SKOV3/CBP cells treated with vehicle, CBP (200 μM), IPN60090 (50 μM), and CBP (200 μM) combined with IPN60090 (50 μM) for 72 h.ROS levels F, and statistical data (mean ± SD) G. Flow cytometry analysis of cell apoptosis rate (mean ± SD) H. Cleaved caspase-3 protein abundance I

Fig. 8
Fig. 8 Patient-derived organoid (PDO) models with ZSWIM4 expression are sensitive to combined therapy.A Immunohistochemical analysis of ZSWIM4 protein expression in EOC tissues from two patients.B, C PDOs were treated with vehicle, CBP (100 μM) combined with PTX (10 nM), IPN60090 (50 μM), or CBP combined with PTX plus IPN60090, and the cell viability was measured on days four and seven using an ATP detection kit.PDO #1 B; PDO#2 C. Typical images (left) and statistical data (mean ± SD) (right).Veh, vehicle.IPN, IPN60090

Fig. 9
Fig.9 Graphic model of ZSWIM4 inhibition enhancing the chemosensitivity of EOC cells Epithelial ovarian cancer (EOC) cells survive in the presence of carboplatin by inducing ZSWIM4 expression via FOXK1-mediated transcription.Elevated ZSWIM4 levels upregulate the glycine biosynthesis-associated rate-limiting enzymes represented by SHMT2 and PHGDH, leading to increased intracellular glycine content and decreased levels of reactive oxygen species, further reducing the sensitivity of EOC cells to chemotherapy.IPN60090, a ZSWIM4-targeting inhibitor, enhances the chemosensitivity of EOC cells.